The extracellular adenosine receptors have a modulatory role in the nervous, circulatory, endocrine and immunological systems. The prospect of harnessing these effects specifically for therapeutic purposes is attractive. We have recently synthesized highly selective A3 adenosine receptor agonists, antagonists, allosteric modulators. A3 agonists are under development for treating cancer, rheumatoid arthritis, and other diseases. Allosteric enhancers promise to be more specific in their action in an affected tissue, than a classical agonist, which can act at all locations of the receptor in the body. Imidazoquinoline and pyridinylisoquinoline derivatives were found to enhance the actions of agonists of the A3 receptor, and thus may prove to be suitable leads for the development of therapeutic agents based on this concept. We have identified two new classes of allosteric modulators of the A3 receptor and are currently exploring the structure activity relationship (SAR). The potential of A3 agonist therapy is of great interest. We are collaborating with Dr. Bruce Liang and Dr. Asher Shainberg on various aspects of the use of adenosine receptor agonists in protection of the heart. We have designed a mixed agonist of A1 and A3 subtypes, both of which are protective in the heart. A mixed A1/A3 agonist is protective in a model of ischemia in skeletal muscle. The adenosine A3 agonist IB-MECA is currently in clinical trials for use in autoimmune inflammatory diseases and cancer conducted by our CRADA partner Can-Fite Biopharma. This compound has already shown clinical efficacy in Phase 2 trials for treatment of rheumatoid arthritis, psoriasis, and dry eye disease. IB-MECA (CF-101) has just entered Phase 2/3 clinical trials for psoriasis. The 2-chloro analogue is currently in clinical trials for liver cancer, and it was shown to reduce viral load in several patients that are infected with hepatitis C virus. Other more selective A3 agonists from our lab, such as the conformationally constrained MRS3558, are of interest for their protective properties. One of the issues in the development of adenosine receptor ligands is the species dependence. Some compounds that are very potent at a given human adenosine receptor are weak in rat tissue. We have developed adenosine agonists and antagonists that work generally across species. The key to A3 receptor ligands that are potent and selective across species is the use of the nucleoside structure as the starting point in the design process. Nucleosides tend to bind to that receptor subtype with greater consistency across species than nonpurine heterocycles. Adenosine A3 antagonists may be useful for the treatment of glaucoma. Early efforts to identify antagonists of the A3 receptor in our library involved screening of chemically diverse libraries. One of the limitations of this approach is that the antagonists often bind well only at the human, but not murine A3 receptors. We are currently developing other novel A3 antagonists based on nucleotide structures, that have proven to be generally applicable across species. We are currently studying systematically the SAR of adenosine derivatives that affect efficacy as A3 adenosine receptor agonists. Surprisingly, a commonly used A1-selective agonist, cyclopentyladenosine, was found to act as a pure antagonist at the A3 subtype. Other nucleosides may be chemically modified, especially on the ribose moiety, to have reduced efficacy at the A3 receptor. Some of these analogues derived from highly potent A3 agonists, such as 5'-truncated nucleosides, were found to be A3 antagonists. Several novel nucleoside-based antagonists of the A3 receptor, including a rigid spirolactam derivative MRS1292 and a truncated 4'-thioadenosine derivative (collaboration with Prof. Lak Shin Jeong, EHWA Univ., Seoul Korea) were found to lower intraocular pressure a mouse model of glaucoma (demonstrated by Prof. Mort Civan, Univ. of Pennsylvania). We are using mutagenesis to study the determinants of recognition of adenosine within the binding site of the A2A and A3 receptors, and proposing conformational factors involved in receptor activation. Since the four subtypes of adenosine receptors have been cloned it has been possible to conduct molecular modeling of the receptor protein, based on sequence analyses and homology modeling using the high resolution rhodopsin structure as template. We intend to use such a modeling approach for the design of more selective adenosine receptor agonists and antagonists. Recently this project has also focused on the effects of adenosine agonists and antagonists in the central nervous system and in the heart and on the possibility of therapeutics for treating neurodegenerative and cardiovascular diseases. An A3 agonist, administered chronically, proved to be highly cerebroprotective in an ischemic model in gerbils. A3 agonists cause morphological and biochemical changes in astroglial cells. Adenosine is released in large amounts during myocardial ischemia and is capable of activating both A1 or A3 receptors that occur on cardiac myocytes to exert a potent cardioprotective effect. We have shown that synthetic adenosine agonists,selective for either the A1 or A3 subtype, protect ischemic cardiac myocytes in culture and in the isolated perfused heart and thus might be beneficial to the survival of the ischemic heart. We recently succeeded in identifying new chemotypes for antagonists of the A2A receptor using structure-based drug discovery (collaboration with B. Shoichet, Univ. of California, San Francisco). We also introduced a fluorescence polarization assay for affinity at the same receptor, that avoids the use fo radioactivity in the drug discovery/screening process. Adenosine receptor agonists, including those selective for the A2A subtype, are in clinical trials for therapeutic and diagnostic applications. Known A2A receptor agonists, most of which contain large substituents at various positions, display anti-inflammatory and vasodilatory properties. We have discovered a means of reducing the size of the ribose moiety of 4-thioadenosine agonists by truncation that preserves the ability to potently activate the A2A receptor. The same modification in nucleosides that are selective for the A3 receptor was shown previously to convert agonists into antagonists at that subtype, but the present series, modified with an A2A receptor-favoring group at the 2 position of the adenine moiety, maintained the ability to fully activate this subtype. Thus, there is a major difference in the mode of activation between the two subtypes. The coexistence of A2A receptor agonism and A3 receptor antagonism in this series of sterically small nucleosides might prove to be beneficial therapeutically in a synergistic manner. Activation of G protein-coupled receptors (GPCRs) upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. In collaboration with the Stevens group, we recently reported the crystal structure of the A2A adenosine receptor bound to an agonist UK-432097 (formerly in clinical trials for COPD) at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A2A receptor, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A2A receptor and its ligand. Our results define the UK-432097 as a conformationally selective agonist capable of receptor stabilization in a specific active state, in contrast to other GPCR agonists that sample multiple conformational states.